Gene transfer preparation

ABSTRACT

A process for producing a gene transfer preparation which comprises adding one or more additives selected from among arginine, glutamic acid or its sodium salt, serine, glucose, inositol, lactose, mannitol, sorbitol, trehalose and xylose to a recombinant virus vector followed by freeze-drying.

TECHNICAL FIELD

This invention relates to a process for producing a freeze-driedpreparation of a virus vector for gene therapy which is excellent insafety and storage stability and a gene transfer preparation obtained bythis process.

BACKGROUND ART

Owing to the rapid progress in genetic engineering, there have beendeveloped various molecular biological processes. With thesedevelopments, techniques for analyzing genetic information and genefunctions have been remarkably advanced. As a result, a number ofattempts have been made to feed back the results thus achieved intoactual clinical treatments. One of the most remarkable advances has beenachieved in the field of gene therapy. That is to say, there have beensuccessfully identified and decoded genes causative of varioushereditary diseases. On the other hand, techniques have been establishedfor physically or chemically transferring these genes into cells.Accordingly, gene therapy has already completed the stage of fundamentalexperiments and thus reached the stage of clinical application.

Since the first clinical test on gene therapy was performed in 1989 inthe United States, gene therapy has been already applied to clinicaltests in Italy, the Netherlands, France, England and China. In theUnited States, in particular, the Recombinant DNA Committee (RAC) of NIHhas approved 54 gene therapy protocols by July 1994 and, therefore,attempts have been made to apply gene therapy to the treatment ofhereditary diseases such as congenial immunological deficiency(adenosine deaminase deficiency), familial hypercholesterolemia andcystic fibrosis and various types of cancer such as malignant melanomaand glioma. Moreover, a number of fundamental studies on the genetherapy for AIDS have been made in recent years.

Gene therapy is classified into germline cell gene therapy and somaticcell gene therapy depending on the type of the target cells to whichgenes are to be transferred. From another point of view, it isclassified into augmentation gene therapy wherein a new (normal) gene isadded while leaving the abnormal (causative) gene as such andreplacement gene therapy wherein the abnormal gene is replaced by thenormal one. At the present stage, the augmentation gene therapy onsomatic cells is exclusively effected due to ethical and technicalrestrictions. A gene therapy process performed today comprises takingout the target cells from the body and, after the completion of the genetransfer, putting the cells back into the body again throughself-transplantation (i.e., ex vivo gene therapy). Further, it is nowunder consideration to administer genes directly to patients in future(i.e., in vivo gene therapy).

One of the large problems in the clinical application of gene therapy ishow to safely and efficiently introduce a foreign gene into the targetcells. Although it was tried to employ physical procedures such asmicroinjection early in the 1980's , only a poor transfer efficiencycould be established and genes could not be transferred in a stablestate thereby. Furthermore, the limited techniques for cell incubationon a mass scale in those days made it impossible to put such attemptsinto practical use. Subsequently, there were developed recombinantviruses (virus vectors) for efficiently transferring foreign genes intotarget cells, which made it possible for the first time to apply thegene therapy to clinical purposes.

There are several types of virus vectors as will be describedhereinbelow. The virus vectors most frequently employed in the genetherapy today are retrovirus vectors originating in moloney murineleukemia virus (MoMLV). That is to say, genes are transferred by takingadvantage in the propagation manner of this virus. A retrovirus is anRNA virus having an envelope which invades into cells through the bondof the envelope protein to the receptor in the host cell side. After theinvasion, the single-stranded virus RNA is converted into adouble-stranded DNA via a reverse transcriptase and thus integrated intothe genomic DNA of the infected cells in a stable state, though atrandom. However, the integration cannot be completed unless the cellsare dividing and proliferating Miller D. G., et al., Molecular andCellular Biology, 10 (8), 4239 (1990)!. The retrovirus gene thusintegrated is called a provirus. From this provirus, RNA is transcribedand thus viral proteins are synthesized. Then new viral particles areformed from these proteins and the virus RNA. In a retrovirus vector,the retrovirus gene in the above-mentioned case recombines with aforeign gene Miller A. D., Current Topics in Microbiology andImmunology, 158, 1 (1992)!. On the MoMLV vector, a number of studieshave been carried out hitherto and many improvements have been achievedon the safety thereof. As a result, no serious trouble has occurred sofar. With respect to the MoMLV vector, however, it is known that thegene is integrated into the genomic DNA of the target cells at randomand the long terminal repeat (hereinafter referred to simply as LTR)sustains the promotion activity for expressing the gene. Therefore, itcannot be denied that the random integration of the foreign gene mighthappen to activate an oncogen existing therearound by chance so as tocause carcinogenesis in the target cells, though there has never beenreported such a case so far. Thus, it has been urgently required todevelop vectors with improved safety. From a practical viewpoint, themost serious problem regarding the MoMLV vector resides in that a genecannot be transferred thereby into cells which are not under division.This fact makes gene repair in neuroblasts impossible in a number ofcongenital metabolic errors. Moreover, hematopoietic stem cells, livercells, muscle cells, etc. to be treated by gene therapy are usually onthe stationary stage in most cases and thus a gene can be transferredthereinto only at a low efficiency. Although cells taken off from thebody are subjected to treatments for promoting division so as to elevatethe gene transfer efficiency, it is seemingly difficult to transfer agene into the above-mentioned cells in vivo. Therefore, it is requiredto develop vectors ensuring efficient gene transfer into cells not beingunder division too in future.

Although herpes virus vectors are expected as being usable in thetransfer of a foreign gene into neuroblasts Palella, T. D., et al., Mol.Cell. Biol., 8, 457, (1988)!, the potent cytotoxicity and large genomicsize (150 kb) disturb the development thereof.

HIV vectors have been developed as vectors which enable specific genetransfer into CD4-positive T lymphocytes owing to the hostcharacteristics of the virus per se Shimada, T., et al., J. Clin.Invest., 88, 1043 (1991)!. Since lymphocytes serve as important targetcells in gene therapy for congenital immunological deficiency, AIDS,cancer, etc., expections are placed on the usefulness of the HIVvectors. The largest disadvantage of the HIV vectors resides in thatthey might be contaminated with wild strains. If this problem could besolved, the HIV vectors might be employed in gene therapy in vivo viaintravascular administration.

Further, adenovirus vectors have attracted public attention, since theyenable gene transfer into cells which are not under division and can beeasily concentrated to a level of about 10¹⁰. Recent studies indicatethat genes can be transferred in vivo at a high ratio into airwayepithelial cells, liver cells, muscular cells, etc. by using theseadenovirus vectors Lavrero, L. D., et. al., Hum. Gene Therapy, 1, 241(1990); Quantin, B., Proc. Natl. Acad. Sci. U.S.A., 89, 2581 (1992)!. Onthe other hand, such an adenovirus vector essentially has acharacteristic that a foreign gene is not integrated into the genomicDNA of the target cells. After treating the target cells with thevector, therefore, the effects of the gene transfer can be sustainedonly for several weeks or several months at the longest. Accordingly, itis required to repeat the gene transfer, which brings about someproblems such as increased physical and mental stress for the patient, adecrease in the gene transfer efficiency due to the appearance ofanti-adenovirus antibody, etc. In addition, clinical attempts have beenalready initiated to administer an adenovirus vector with a bronchoscopefor treating cystic fibrosis. However, it is reported that inflammatoryresponses arise in these cases due to the immunogenicity andcytotoxicity of the adenoviral particles.

In contrast, adeno-associated virus (AAV) vectors are characterized inthat a foreign gene is integrated into the genomic DNA of the targetcells and the vectors have neither any pathogenicity nor cytotoxicityMuzyczka, N., Currnet Topics in Microbiology and Immunology, 158, 97(1992)!. Moreover, the ITR (inverted terminal repeat) thereof, which isneeded in packaging viral particles and gene integration into genomicDNA, has no promoting activity for gene expression. Thus, the geneexpression can be arbitrarily switched on/off by setting an appropriateinner promoter or a tissue-specific promoter can be employed. In thecase of the AAV vectors, use can be made of hosts over a wide range,which makes these vectors applicable to various target cells/diseases.Owing to these characteristics, the AAV vectors are expected as novelvirus vectors, i.e., a substitute for the MoMLV vectors. It is alsofound that AAV of wild type is integrated into a definite site in the19th chromosome Suwadogo, M. and Roeder, R. G., Prc. Natl. Acad. Sci.U.S.A, 82, 4394 (1985)!. Thus AAV vectors attract public attention asvectors capable of targeting the gene integration site.

However, any manufacturing pharmaceutical discussion has been made onnone of these virus vectors in order to storage them in a stable stateand maintain the uniformity thereof. Although virus vectors are storedin a frozen state today, the storage period is limited and it isobserved that the virus vectors suffer from a decrease in titer with thepassage of time. In practical clinical studies, it is therefore neededto prepare a vector in each test and examine the decrease in the genetransfer efficiency during the storage prior to the treatment. Sincesuch examinations comprise complicated procedures and take aconsiderably long time, it has been strongly required to establish amethod for supplying stabilized virus vectors having improved anduniform performance.

It was attempted to freeze-dry MoMLV vectors by using gelatin as astabilizer Kotani, H., et al., Human Gene Therapy, 5, 19 (1994)!. Sincegelatin usually originates in animals such as swine, it might serve asan immunogen at a high possibility when administered in vivo. Thus, theabove method cannot be always referred to as a safe one.

In clinical studies on gene therapy performed today, detailedexaminations are made on the type of vectors and the pharmacologicaleffects of genes for therapeutic use. Because they are preparations forgene therapy, virus vectors should be supplied safely so as to ensure auniform performance. Namely, it is essentially required to establish aprocess for storing these vectors in a stable state. However, fewstudies have been made in this field.

DISCLOSURE OF THE INVENTION

The present inventors have conducted extensive studies to solve theabove-mentioned problems. As a result, they have successfullyestablished a technique for stably supplying virus vectors having highsafety and uniform performance, i.e., a technique for storing virusvectors in a stable state, thus making it possible to freeze-dry variousvirus vectors without lowering the gene transfer efficiency.

It is known that several types of viruses would not lose theirinfectivity even after freeze-drying. In such a case, it has been apractice to add gelatin and saccharides thereto. On the other hand,attempts have been also made to freeze-dry virus vectors similar tothese viruses. In these cases, gelatin and saccharides are added too.However, it cannot always be considered as a safe method, since therearises a fear that these additives might serve as an immunogen at a highpossibility when administered in vivo.

Under these circumstances, it has been attempted in the presentinvention to develop a freeze-drying method whereby a high gene transferefficiency can be sustained not by using gelatin, etc. which might serveas an immunogen but by using exclusively low-molecular-weight substanceswhich have been already employed as pharmaceutical additives.

Accordingly, the present invention provides a process for producing agene transfer preparation which comprises adding one or more additivesselected from among arginine, glutamic acid or its sodium salt, serine,glucose, inositol, lactose, mannitol, sorbitol, trehalose and xylose toa recombinant virus vector followed by freeze-drying.

The present invention further provides a gene transfer preparationproduced by the above-mentioned process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which shows gene transfer efficiencies achieved byadding various additives each at a concentration of 5%.

FIG. 2 is a graph which shows gene transfer efficiencies achieved byadding various additives each at concentrations of 2.5% and 5%.

FIG. 3 is a graph which shows gene transfer efficiencies achieved byadding combinations of two additives each at a concentration of 2.5%.

BEST MODE FOR CARRYING OUT THE INVENTION

The virus vector to be used in the present invention may be an arbitraryone usable in gene therapy selected from among, for example, theabove-mentioned moloney murine leukemia virus (MoMLV) vectors, herpesvirus vectors, adenovirus vectors, adeno-associated virus vectors andhuman immunodeficiency virus (HIV) vectors. Such a vector is dissolvedin a medium such as DMEM medium or PBS to thereby give a virus vectorstock solution. The virus vector stock solution may have an arbitraryconcentration.

The process of the present invention can be achieved by addingadditive(s) to the above-mentioned virus vector stock solution andfreeze-drying the same. It is preferable that the additives to be usedin the present invention are less immunogenic substances such aslow-molecular-weight amino acids, derivatives thereof, saccharides andderivatives thereof.

Preferable examples of the amino acids and derivatives thereof includearginine, glutamic acid or its sodium salt and serine. Among all,glutamic acid or its sodium salt is particularly preferable therefor.

Preferable examples of the saccharides and derivatives thereof includeglucose, inositol, lactose, mannitol, sorbitol, trehalose and xylose.Among all, glucose is the most desirable example thereof.

One or more substances selected from these amino acids and saccharidesmay be freely combined and employed as the additive(s) depending on thetype of the virus vector employed, the concentration of the virus vectorstock solution, etc. Selection can be made of a combination of an aminoacid with another amino acid, a saccharide with another saccharide, oran amino acid with a saccharide. Among all, the combination of sodiumglutamate with glucose is the most desirable one, since a high genetransfer efficiency (virus vector titer) can be maintained thereby.

Each additive selected from amino acids and saccharides is used at aweight ratio to the vector solution of from about 1% to about 10%,preferably from about 1.5% to about 7% and still preferably from about1.5% to about 5%.

The solution may further contain ascorbic acid, polyethylene glycol,polyvinylpyrrolidone, polyvinyl alcohol, preservatives, etc. Since it ispreferable that the virus vector solution is isotonic, the osmoticpressure of the solution can be controlled by adding a buffer thereto.

The thus obtained virus vector solution containing various additives isfreeze-dried. Freeze-drying can be performed by a publicly known method.For example, the virus vector solution is frozen with liquid nitrogenand then treated with a freeze-dryer (manufactured by Finaqua). Thefreeze-dried gene transfer preparation was then packed in vials andstored preferably at low temperatures till using. The gene transferpreparation of the present invention can be reconstituted with waterbefore using. As will be shown in the Examples hereinafter, the virusvector reconstituted with water sustained a high gene transferefficiency.

The present invention makes it possible to obtain a gene transferpreparation sustaining a high gene transfer efficiency by using not anyingredient likely to serve as an immunogen (gelatin, etc.) butexclusively low-molecular-weight substances which have been alreadyemployed as pharmaceutical additives. The gene transfer preparation ofthe present invention can be easily stored and sustains a high titer.Therefore, it is usable in every virus vector preparation and has a verybroad application range.

EXAMPLES

To further illustrate the present invention in greater detail, and notby way of limitation, the following Examples will be given.

Example 1

Preparation of recombinant MoMLV vector

A cell culture dish (9 cm in diameter) was inoculated with PA317/β-19(provided by Prof. Shimada, Nippon Medical School) capable of producinga recombinant MoMLV vector containing neomycin resistance gene. Thenthese cells were incubated in DMEM medium (manufactured by Gibco)containing 10% of fetal calf serum (manufactured by Gibco) under usualconditions (37° C., 5% CO₂) up to about 80% confluence. Subsequently,the medium was replaced and, 12 hours thereafter, the medium containingthe recombinant MoMLV vector was recovered and referred to as the virusvector stock solution.

Example 2

Preparation and freeze-drying of recombinant MoMLV vector solution

To the virus vector stock solution obtained in Example 1 were added theamino acids, saccharides or combinations thereof as given in FIGS. 1 to3 in such a manner as to give a final concentration of 5% or 2.5%. Afterfreezing with liquid nitrogen, each sample was freeze-dried with afreeze-dryer (manufactured by Finaqua) over day and night. Thefreeze-dried product thus obtained was stored at -40° C. till using. Asa control, an additive-free sample was also prepared. Each additive wasa product manufactured by Wako Pure Chemical Industries, Ltd.

Example 3

Method for determining the titer (gene transfer efficiency) ofrecombinant MoMLV vector

A cell culture dish (6 cm in diameter) was inoculated with 3T3 cells(manufactured by Dainippon Pharmaceutical Co., Ltd.). These cells wereincubated in DMEM medium (manufactured by Gibco) containing 10% of fetalcalf serum (manufactured by Gibco) under usual conditions (37° C., 5%CO₂) up to 80% confluence and then employed in the determination of thetiter.

Distilled water for injection (manufactured by Otsuka PharmaceuticalCo., Ltd.) was added to each of the freeze-dried products obtained inExample 2 to thereby prepare a re-suspension of the vector having thesame volume as the one before freeze-drying. 10 μl of this re-suspensionof the vector was mixed with 990 μl of DMEM containing 10% of fetal calfserum to thereby give a vector solution for titer determination. Fromthe 3T3 cells incubated up to 80% confluence, the medium was eliminatedand 1,000 μl of the vector solution for titer determination was addedthereto. After incubating the cells under the usual conditions for 4hours, 3 ml of DMEM containing 10% of fetal calf serum was added theretoand the incubation was continued for additional 24 hours. Subsequently,the cells were incubated in DMEM containing 10% of fetal calf serumcontaining 800 μg/ml of G418 (manufactured by Gibco), i.e., an analog ofneomycin. The number of the drug-resistant colonies thus formed wasreferred to as the titer (cfu/ml).

Example 4

Determination of the titer of recombinant MoMLV vector

FIG. 1 shows the results obtained by using various additives each at aconcentration of 5%. Thus, it is revealed that glucose, sodiumglutamate, mannitol and trehalose achieved high titers. FIGS. 2 and 3show the results obtained by adding these additives each at aconcentration of 2.5% and using combinations of two additives. Theseresults indicate that a freeze-dried virus vector preparation with ahigh titer can be obtained by using sodium glutamate with glucose.

We claim:
 1. A process for producing a gene transfer preparation whichcomprises adding an additive, which is a combination of glutamic acid orits sodium salt with glucose, to a recombinant virus vector followed byfreeze-drying.
 2. A process as claimed in claim 1, wherein eachcomponent of the additive is used at a weight ratio to the vectorsolution of from about 1% to about 10%.
 3. A gene transfer preparationproduced by a process as claimed in claim 1.